JP5681740B2 - Method for adjusting expression of hydroxymethylglutaryl CoA reductase by bHLH transcription factor, isoprenoid production plant introduced with gene encoding bHLH transcription factor, and method for producing polyisoprenoid using the isoprenoid production plant - Google Patents

Description

The present invention relates to a method for regulating the expression of hydroxymethylglutaryl CoA reductase using a bHLH transcription factor, an isoprenoid production plant into which a gene encoding a bHLH transcription factor has been introduced, and production of a polyisoprenoid using the isoprenoid production plant Regarding the method.

Natural rubber (a type of polyisoprenoid) currently used in industrial rubber products should be collected from rubber-producing plants such as Hevea brasiliensis and Mulberry plant Ficus elastica. Is obtained.

At present, the natural rubber used in industrial rubber products is almost exclusively collected from para rubber tree. Para rubber tree is a plant that can grow only in limited areas such as Southeast Asia and South America. Furthermore, it takes about 7 years for para rubber trees to become an adult tree from which rubber can be collected, and the period during which natural rubber can be collected is limited to 20-30 years. In the future, demand for natural rubber is expected to increase mainly in developing countries. However, for the reasons described above, it is difficult to significantly increase the production of natural rubber using para rubber tree. For this reason, there is a concern about the depletion of natural rubber resources, and there is a demand for improving the natural rubber production efficiency in Para rubber tree, as well as a stable natural rubber supply source other than Para rubber tree.

Examples of a method for improving the production efficiency of natural rubber in Para rubber tree include a method of collecting a larger amount of latex in order to obtain a larger amount of natural rubber. Specifically, for example, a method of adding stimulation to the trunk of rubber tree with ethylene or Etherephone (2-chloroethylphosphonic acid), lanolin containing jasmonic acid or linolenic acid of jasmonic acid precursor, etc. There is a method of promoting (for example, Non-Patent Document 1).

However, the method of increasing latex production by ethylene stimulation may cause cracking of the bark if it is used for a long period of time on the trunk. In addition, ethylene stimulation is a method that makes the latex outflow from the milk duct more smooth, and does not improve the latex production capacity of the tree itself. .

It is also possible to increase the number of milk ducts by using jasmonic acid or the like to increase the number of milk ducts, but when collecting latex by tapping, the latex flowing out from the milk duct coagulates at the wound and is produced. There is a problem that the collected latex cannot be recovered sufficiently.

On the other hand, it is known that the biosynthetic amount of polyisoprenoid varies depending on the light response, injury response, cold treatment, and the like. However, it is not specifically known which transcription factor is activated by these responses and controls the amount of polyisoprenoid biosynthesis.

Hao, et al., Anals of Botany, 2000, 85, 37-43.

An object of the present invention is to solve the above-mentioned problems and to provide a method for enhancing the biosynthetic pathway of polyisoprenoid. It is another object of the present invention to provide an isoprenoid-producing plant with enhanced polyisoprenoid biosynthetic pathway and a method for producing polyisoprenoid using the isoprenoid-producing plant.

The present invention relates to a method for regulating the expression of hydroxymethylglutaryl CoA reductase with a basic-helix-loop-helix type transcription factor (bHLH type transcription factor).

In the above method, it is preferable to adjust the expression of the hydroxymethylglutaryl CoA reductase in the host by introducing a gene encoding the bHLH transcription factor into the host.

The gene is preferably a DNA described in [1] or [2] below.
[1] DNA comprising the base sequence represented by any one of SEQ ID NOs: 1, 3 and 5
[2] A protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 1, 3, and 5 and has a transcription factor activity DNA encoding

More preferably, the method is a method for enhancing the expression of hydroxymethylglutaryl CoA reductase.

The bHLH transcription factor is preferably a protein according to any one of [1] to [3] below.
[1] A protein comprising the amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6 [2] In the amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6 Or a protein comprising a sequence containing a plurality of amino acid substitutions, deletions, insertions, and / or additions and having transcription factor activity [3] represented by any one of SEQ ID NOs: 2, 4, and 6 A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence and having transcription factor activity

The host is preferably an isoprenoid-producing plant.

The present invention also relates to an isoprenoid-producing plant into which a gene encoding a bHLH transcription factor has been introduced.

The gene is preferably a DNA described in [1] or [2] below.
[1] DNA comprising the base sequence represented by any one of SEQ ID NOs: 1, 3 and 5
[2] A protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 1, 3, and 5 and has a transcription factor activity DNA encoding

The present invention also relates to a method for producing a polyisoprenoid using the above isoprenoid-producing plant.

The method of the present invention enables the expression of hydroxymethylglutaryl CoA reductase, a rate-limiting step of the mevalonate (MVA) pathway located upstream of the polyisoprenoid biosynthetic pathway, to express the basic-helix-loop-helix type transcription factor (bHLH type). Therefore, the biosynthetic pathway of polyisoprenoids can be suitably enhanced by enhancing the rate-limiting site when isopentenyl diphosphate (IPP) is biosynthesized by the MVA pathway. In addition, since the isoprenoid-producing plant of the present invention is an isoprenoid-producing plant into which a gene encoding a bHLH transcription factor has been introduced, the polyisoprenoid biosynthetic pathway is suitably enhanced, and the isoprenoid-producing plant is used. By producing polyisoprenoid, the production amount of polyisoprenoid can be increased.

It is a schematic diagram which shows a part of biosynthetic pathway of a polyisoprenoid.

The present inventors conducted various studies in order to enhance the biosynthetic pathway of polyisoprenoid. A schematic diagram showing a part of the biosynthetic pathway of polyisoprenoid is shown in FIG. Among the biosynthetic pathways of polyisoprenoids, the mevalonic acid (MVA) pathway (Cytosol MVA pathway shown in FIG. 1) and MEP are used as a pathway for biosynthesizing one of the important substances, isopentenyl diphosphate (IPP). Two types of routes (Plastical DXP pathway described in FIG. 1) are known.

The present inventors pay attention to the MVA pathway because it is a general pathway for providing IPP in rubber latex, and consider the enhancement of the pathway surrounded by the dotted line in FIG. A plurality of proteins that are expected to play an important role are selected from the various proteins involved in the protein. Among them, hydroxymethylglutaryl CoA reductase (HMG-CoA reductase) is MVA. We found that it was the rate-limiting step of the route.

The present inventors then searched for a transcription factor capable of controlling the expression of HMG-CoA reductase in order to control the expression of HMG-CoA reductase. Specifically, a gene encoding the HMG-CoA reductase derived from the leaves of Para rubber tree (the amino acid sequence of the HMG-CoA reductase is shown in SEQ ID NO: 8 in the sequence listing) (the nucleotide sequence of the gene is shown in SEQ ID NO: 7 in the sequence listing) And a DNA fragment containing the promoter region were cloned (for details, see Examples). Then, the nucleotide sequence of the obtained DNA fragment was investigated, and the nucleotide sequence of the promoter region of the gene encoding HMG-CoA reductase was clarified.

Furthermore, the nucleotide sequence of the clarified promoter region was analyzed using an analysis database for plant promoters (A Date of Plant Cis-acting Regulatory DNA Elements (PLACE)). As a result of the analysis, many binding (recognition) sites (CANNTG) of basic-helix-loop-helix type transcription factor (bHLH type transcription factor) were found.

From this analysis result, the bHLH transcription factor is a transcription factor capable of controlling the expression of HMG-CoA reductase, that is, the bHLH transcription factor can enhance the rate-determining site of IPP biosynthesis by the MVA pathway, and is a polyisoprenoid. It was strongly suggested that it is a transcription factor that can suitably control the biosynthetic pathway. When a confirmation test was performed using yeast, among the bHLH transcription factors, Myc2 (the base sequence and amino acid sequence of Myc2 derived from Arabidopsis thaliana are shown in SEQ ID NOs: 1 and 2, respectively) , ILR3 (The base sequence and amino acid sequence of ILR3 derived from Arabidopsis thaliana are shown in SEQ ID NOs: 3 and 4, respectively), HFR1 (The base sequence and amino acid sequence of HFR1 derived from Arabidopsis thaliana are shown in SEQ ID NOs: 5 and 6, respectively) It was confirmed that the use of can enhance the expression of HMG-CoA reductase.

Thus, the present inventors have shown that bHLH type transcription factor is a transcription factor that can suitably enhance the expression of HMG-CoA reductase, can enhance the rate-limiting site of IPP biosynthesis by MVA pathway, and can produce polyisoprenoids. It was discovered that the transcription factor can suitably enhance the synthetic pathway. Then, the bHLH transcription factor enhances the rate-limiting site of IPP biosynthesis by the MVA pathway, whereby the polyisoprenoid biosynthesis pathway can be suitably enhanced, and the gene encoding the bHLH transcription factor is It has been found that the amount of polyisoprenoid produced can be increased by introducing it into an isoprenoid-producing plant and producing the polyisoprenoid using the isoprenoid-producing plant.

In the present specification, hydroxymethylglutaryl CoA reductase (HMG-CoA reductase) is one of the rate-limiting enzymes of the mevalonate pathway, and hydroxymethylglutaryl CoA reductase (NADPH) (EC 1.1.1. 34) and hydroxymethylglutaryl CoA reductase (EC 1.1.1.88).

The method of the present invention is a method of regulating the expression of hydroxymethylglutaryl CoA reductase using a bHLH transcription factor.

The bHLH transcription factor is not particularly limited as long as it is a transcription factor having a structural feature of a basic helix-loop-helix motif. For example, Myc2 (the base sequence and amino acid sequence of Myc2 derived from Arabidopsis 1 and 2), ILR3 (the base sequence and amino acid sequence of ILR3 derived from Arabidopsis thaliana are shown in SEQ ID NO: 3 and 4, respectively), HFR1 (the base sequence and amino acid sequence of HFR1 derived from Arabidopsis thaliana are shown in SEQ ID NO: SEQ ID NO. 5 and 6), and the like.
In the present specification, a transcription factor refers to a protein having an activity of increasing or decreasing (preferably increasing) the amount of gene transcription.

The origin of the bHLH transcription factor is not particularly limited, but is preferably a transcription factor derived from Para rubber tree, Nogeshi, Guayule, Russian dandelion, or Arabidopsis thaliana.

(Amino acid sequence of bHLH transcription factor)
Specific examples of bHLH transcription factors include [1] below.
[1] A protein comprising the amino acid sequence represented by any one of SEQ ID NOs: 2, 4, and 6

Further, it is known that a transcription factor may have transcription factor activity even when it contains substitution, deletion, insertion, or addition of one or more amino acids in the original amino acid sequence. Accordingly, specific examples of bHLH transcription factors include [2] below.
[2] The amino acid sequence represented by any one of SEQ ID NOs: 2, 4 and 6, consisting of a sequence containing one or more amino acid substitutions, deletions, insertions, and / or additions, and transcription Protein having Factor Activity In this specification, transcription factor activity refers to an activity that increases or decreases (preferably increases) the transcription amount of a gene encoding hydroxymethylglutaryl CoA reductase.

In order to maintain the transcription factor activity, in the amino acid sequence represented by SEQ ID NO: 2, preferably one or a plurality of amino acids, more preferably 1 to 119 amino acids, still more preferably 1 to 89 amino acids. Amino acids, particularly preferably 1-60 amino acids, most preferably 1-30 amino acids, more preferably 1-12 amino acids, and most preferably 1-6 amino acid substitutions, deletions, insertions And / or an amino acid sequence containing an addition.

In order to maintain the transcription factor activity, in the amino acid sequence represented by SEQ ID NO: 4, preferably one or a plurality of amino acids, more preferably 1 to 47 amino acids, still more preferably 1 to 35 amino acids. Amino acids, particularly preferably 1 to 23 amino acids, most preferably 1 to 12 amino acids, more preferably 1 to 5 amino acids, and most preferably 1 to 2 amino acid substitutions, deletions and insertions And / or an amino acid sequence containing an addition.

In order to maintain the transcription factor activity, in the amino acid sequence represented by SEQ ID NO: 6, preferably one or a plurality of amino acids, more preferably 1 to 58 amino acids, still more preferably 1 to 44 amino acids. Amino acids, particularly preferably 1 to 29 amino acids, most preferably 1 to 15 amino acids, more preferably 1 to 6 amino acids, and most preferably 1 to 3 amino acid substitutions, deletions, insertions And / or an amino acid sequence containing an addition.

As an example of amino acid substitution, conservative substitution is preferable, and specifically, substitution within a group in parentheses below can be mentioned. For example, (glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

Amino acid substitutions, deletions, insertions, and / or additions are introduced into regions other than the active domain as a bHLH transcription factor, the binding domain to the transcription factor binding site, and other parts important for transcription factor activity. It is preferable. Such a domain can be appropriately determined by those skilled in the art from homology analysis with known bHLH transcription factors.

It is also known that a protein having an amino acid sequence having a high sequence identity with the amino acid sequence of a transcription factor may have the same activity. Accordingly, specific examples of bHLH transcription factors include [3] below.
[3] A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 2, 4 and 6 and having transcription factor activity

In order to maintain transcription factor activity, the sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 2, 4 and 6 is preferably 85% or more, more preferably 90% or more. More preferably, it is 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

The sequence identity of amino acid sequences and base sequences is determined by the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol. , 183, 63 (1990)].

Examples of a method for confirming that the protein has transcription factor activity include, for example, a conventionally known method such as gel shift assay, and reporter assay using β-galactosidase, luciferase, GFP (Green Fluorescent Protein) or the like as a reporter gene. The method of confirming by etc. is mentioned.

In addition, as bHLH type transcription factor,
[1-1] Protein consisting of the amino acid sequence represented by any one of SEQ ID NOs: 2 and 4 [2-1] In the amino acid sequence represented by any one of SEQ ID NOs: 2 and 4, Alternatively, a protein [3-1] consisting of a sequence comprising a plurality of amino acid substitutions, deletions, insertions and / or additions and having transcription factor activity is represented by any one of SEQ ID NOs: 2 and 4 It is preferably any one of proteins having an amino acid sequence having 80% or more sequence identity with the amino acid sequence and having transcription factor activity,
[1-2] Protein consisting of the amino acid sequence represented by SEQ ID NO: 2 [2-2] Substitution, deletion, insertion and / or substitution of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 2 A protein comprising a sequence containing an addition and having transcription factor activity [3-2] A protein having an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having transcription factor activity It is more preferable that it is either.

(DNA encoding bHLH transcription factor)
Examples of DNA encoding a bHLH transcription factor include [1] or [2] below.
[1] DNA comprising the base sequence represented by any one of SEQ ID NOs: 1, 3 and 5
[2] A protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 1, 3, and 5 and has a transcription factor activity DNA encoding

The term “hybridize” as used herein refers to a step in which DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific nucleotide sequence or a part of the DNA sequence is useful as a probe for Northern or Southern blot analysis, or can be used as an oligonucleotide primer for PCR (Polymerase Chain Reaction) analysis. It may be DNA. Examples of DNA used as a probe include DNA of at least 100 bases, preferably 200 bases or more, more preferably 500 bases or more, but may be DNA of at least 10 bases, preferably 15 bases or more. .

Methods of DNA hybridization experiments are well known, for example, Molecular Cloning 2nd Edition, 3rd Edition (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual. In addition to those described in Molecular), hybridization conditions can be determined and experiments can be performed according to a number of other standard textbooks.

The above stringent conditions include, for example, a filter in which DNA is immobilized and probe DNA, 50% formamide, 5 × SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6). ) After incubation overnight at 42 ° C. in a solution containing 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / l denatured salmon sperm DNA, for example 0.2 × SSC solution at about 65 ° C. While the conditions for washing the filter can be raised, lower stringent conditions can also be used. Stringent conditions can be changed by adjusting the concentration of formamide (the lower the formamide concentration, the lower the stringency), and changing the salt concentration and temperature conditions. As low stringent conditions, for example, 6 × SSCE (20 × SSCE is 3 mol / l sodium chloride, 0.2 mol / l sodium dihydrogen phosphate, 0.02 mol / l EDTA, pH 7.4), 0 After overnight incubation at 37 ° C. in a solution containing 5% SDS, 30% formamide, 100 μg / l denatured salmon sperm DNA, 50 ° C. 1 × SSC, 0.1% SDS solution The conditions for using and washing can be raised. Further, examples of lower stringent conditions include conditions under which hybridization is performed using a solution having a high salt concentration (for example, 5 × SSC) under the above-described low stringency conditions and then washed.

The various conditions described above can also be set by adding or changing a blocking reagent used to suppress the background of hybridization experiments. The addition of the blocking reagent described above may be accompanied by a change in hybridization conditions in order to adapt the conditions.

As DNA that can hybridize under the above stringent conditions, for example, when calculated based on the above parameters using a program such as BLAST and FASTA, any one of SEQ ID NOs: 1, 3, and 5 A DNA comprising a nucleotide sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, particularly preferably 99% or more of the nucleotide sequence represented by I can give you.

As a method for confirming that the DNA that hybridizes with the above-described DNA under stringent conditions is a DNA encoding a protein having transcription factor activity, for example, a conventionally known method such as gel shift assay, β -The method of confirming by the reporter assay etc. which used galactosidase, luciferase, or GFP (Green Fluorescent Protein) etc. as a reporter gene is mentioned.

In addition, as DNA encoding bHLH type transcription factor,
[1-1] DNA comprising the base sequence represented by any one of SEQ ID NOs: 1 and 3
[2-1] A protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 1 and 3 and has transcription factor activity. DNA encoding
It is preferable that either
[1-2] DNA consisting of the base sequence represented by SEQ ID NO: 1
[2-2] DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and encodes a protein having transcription factor activity
It is more preferable that it is either.

In addition, bHLH type transcription factor and DNA encoding the bHLH type transcription factor are described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbolo Laboratory Press (1989), Current Proportional Health Press (1989), Current Protocol. 1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985) etc., using the site-directed mutagenesis method described in, for example, the base sequence represented by any one of SEQ ID NOs: 1, 3, and 5 (base sequence of Myc2 derived from Arabidopsis thaliana) It can be obtained by introducing a site-specific mutation into the base sequence of ILR3 derived from Arabidopsis thaliana or the base sequence of HFR1 derived from Arabidopsis thaliana.

(Transformant)
By introducing a gene encoding a bHLH transcription factor into a host, an organism (transformant) transformed to express the bHLH transcription factor can be obtained. In the transformant, the expression of hydroxymethylglutaryl CoA reductase is regulated (enhanced) by expressing the bHLH transcription factor. As a result, the rate-limiting site of IPP biosynthesis by the MVA pathway is enhanced, whereby the polyisoprenoid biosynthesis pathway is suitably enhanced, and the amount of polyisoprenoid produced in the transformant can be suitably increased.

Next, a method for producing an organism (transformant) transformed so as to express a bHLH transcription factor will be briefly described. Here, a method for producing a transformant transformed so as to mainly express the bHLH transcription factor will be briefly described. Such a transformant can be prepared by a conventionally known method once the gene encoding the bHLH transcription factor to be introduced is determined.

Specifically, for example, downstream of the promoter of an appropriate expression vector, the base sequence represented by any one of SEQ ID NOs: 1, 3 and 5 (the base sequence of Myc2 derived from Arabidopsis thaliana, the ILR3 derived from Arabidopsis thaliana) A DNA containing a base sequence or a base sequence of HFR1 derived from Arabidopsis thaliana is inserted using an appropriate restriction enzyme or the like to produce a recombinant DNA. A transformant can be obtained by introducing the recombinant DNA into a host cell suitable for the expression vector.

In the above description, the base sequence represented by any one of SEQ ID NOs: 1, 3 and 5 (base sequence of Myc2 derived from Arabidopsis thaliana, base sequence of ILR3 derived from Arabidopsis thaliana, or base sequence of HFR1 derived from Arabidopsis thaliana ) Has been described, but other bHLH transcription factors derived from Arabidopsis thaliana or DNAs encoding bHLH transcription factors derived from organisms other than Arabidopsis thaliana may be used. In this case, the DNA encoding the bHLH transcription factor can be identified and isolated by a known technique, for example, by screening using a part of the base sequence represented by SEQ ID NO: 1 as a probe. That's fine. A method for isolating a target DNA molecule using the DNA molecule as a probe is described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989) and the like. Moreover, you may use the DNA which introduce | transduced the mutation into the said DNA.

As the host (host cell), any microorganisms, yeasts, animal cells, insect cells, plant cells, etc. can be used as long as they can express the gene of interest. Currently, polyisoprenoids are biosynthesized. Since only living organisms are plants (isoprenoid producing plants), plants (isoprenoid producing plants) are preferable as hosts, and plant cells (plant cells of isoprenoid producing plants) are preferred as host cells. If the polyisoprenoid can be biosynthesized in plants other than plant cells as the technology develops in the future, a gene encoding the above bHLH transcription factor may be suitably introduced into the cells. Is possible.

The isoprenoid-producing plant is not particularly limited as long as it is a plant capable of producing isoprenoids. For example, the genus of Hevea such as Hevea brasiliensis; Sonchus oleraceus, Sonchus asper, Hachijouna etc. of Sonchus spp;.. goldenrod (Solidago altissima), goldenrod (. Solidago virgaurea subsp asiatica), Miyama goldenrod (. Solidago virgaurea subsp leipcarpa), tung moth Mine goldenrod (Solidago virgaurea subsp leipcarpaf pal ... Dosa), giant goldenrod (Solidago virgaurea subsp gigantea), Solidago gigantea (Solidago gigantea Ait var leiophylla Fernald) Solidago genus and the like; sunflower (Helianthus annuus), Shirota et sunflower (Helianthus argophyllus), Helianthus Atororubensu (Helianthus atrorubens), Helianthus genus such as sunflower (Helianthus debilis), kohimawari (Helianthus decapetalus), giant sunflower (Helianthus giganteus); dandelion (Taraxacum), ezotapo Po (Taraxacum venustum H.Koidz), Shinano dandelion (Taraxacum hondoense Nakai), Kanto dandelion (Taraxacum platycarpum Dahlst), Kansai dandelion (Taraxacum japonicum), dandelion (Taraxacum officinale Weber), Taraxacum spp such as Russian dandelion (Taraxacum koksaghyz); Figs (Ficus carica), Indian rubber tree (Ficus elastica), Green sea bream (Ficus pumila L.), Inubiwa (Ficus erecta Thumb.), Hosobamuku Inubiwa (Ficus ampelas Burm.). f. ), Chinese beetle (Ficus)
bengenetensis Merr. ), Mikuinubiwa (Ficus irisana Elm.), Ganju (Ficus microcarpa L.f.), Ouineu Niwa (Ficus septica Burm.f.), the genus P. genus P. Examples include the genus Parthenium such as rhinoceros (Parthenium hysterophorus), ragweed (Parthenium hysterophorus); Among them, it is preferably a plant belonging to at least one genus selected from the group consisting of the genus Hevea, Sonchus, Taraxacum, and Parhenium, and selected from the group consisting of Para rubber tree, Nogeshi, Guayule, and Russian dandelion It is more preferable that it is at least one kind.

As the expression vector, one that can autonomously replicate in the host cell or can be integrated into a chromosome and that contains a promoter at a position where the recombinant DNA can be transcribed can be used.

When plant cells are used as host cells, examples of expression vectors include pBI vectors, pUC vectors, Ti plasmids, tobacco mosaic virus vectors, and the like.
Any promoter may be used as long as it functions in plant cells. For example, cauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promoter, nopaline synthase gene promoter, and tobacco mosaic virus 35S. Examples thereof include promoters and rice-derived actin gene promoters.

In addition, it is preferable to use an expression vector having a promoter that is specifically expressed in a tissue in which an isoprenoid compound is biosynthesized, such as a milk duct. By specifically expressing the polyisoprenoid in the biosynthesized tissue, it is possible to suppress harmful effects such as delayed growth of plants.

As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, a method using Agrobacterium (Japanese Patent Application Laid-Open No. 59-140885, Japanese Patent Application Laid-Open No. 59-14088). Sho 60-70080, WO 94/00977), electroporation method (Japanese Patent Laid-Open No. 60-251887), method using particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813) No.) etc.

By the above method or the like, a transformant (transformed plant cell) into which a gene encoding a bHLH transcription factor has been introduced is obtained.

The present invention provides an isoprenoid-producing plant into which a gene encoding a bHLH transcription factor has been introduced. The isoprenoid-producing plant is not particularly limited as long as it is an isoprenoid-producing plant having transformed plant cells. For example, not only transformed plant cells obtained by the above-described method, but also their progeny or clones, and further subculture them. It is a concept that includes all of the progeny plants obtained. Once a transformed plant cell into which the above DNA or vector has been introduced into the genome is obtained, offspring or clones can be obtained from the transformed plant cell by sexual reproduction, asexual reproduction, tissue culture, cell culture, cell fusion, etc. It is possible to obtain. In addition, a propagation material (eg, seed, fruit, cut ear, tuber, tuber, strain, adventitious bud, adventitious embryo, callus, protoplast, etc.) is obtained from the transformed plant cell or its progeny or clone. The isoprenoid-producing plant can be mass-produced.

A method for regenerating plant bodies from transformed plant cells is, for example, the method of Toi et al. (Japanese Patent Application No. 11-127005) in Eucalyptus, and the method of Fujimura et al. (Fujimura et al. (1995), Plant Tissue Culture Lett. , Vol. 2: p74-), for corn, the method of Shirrito et al. (Shillito et al. (1989), Bio / Technology, vol. 7: p581-), for potato, the method of Visser et al. (Visser et al. (1989), Theor. Appl. Genet., Vol. 78: p589-), and in Arabidopsis, the method of Akama et al. (Akama et al. (1992), Plant Cell Rep., Vol. 12: p7-) is known. Regenerating plants from the transformed plant cells with reference to these.

In the regenerated plant body, the expression of the target transcription factor gene can be confirmed by using a well-known technique. For example, the expression of the target transcription factor may be analyzed by Western blot.

As a method for obtaining seeds from the above transformed plant body, for example, the transformed plant body is rooted in an appropriate medium, and the rooted body is transplanted into a pot containing water-containing soil. The seeds are obtained by growing them under appropriate cultivation conditions and finally forming seeds. In addition, as a method for obtaining a plant from seeds, for example, a plant is obtained by sowing seeds derived from a transformed plant obtained as described above in water-containing soil and growing them under appropriate cultivation conditions. You can get a body.

In the present invention, the amount of polyisoprenoid produced can be increased by producing a polyisoprenoid using an isoprenoid-producing plant into which a gene encoding a bHLH transcription factor has been introduced. Specifically, transformed plant cells obtained by the above method, callus obtained from transformed plant cells, cells re-differentiated from the callus, etc. are cultured in an appropriate medium or regenerated from transformed plant cells. The polyisoprenoid can be produced by growing the transformed plant body, the plant body obtained from the seed obtained from the transformed plant body, and the like under suitable cultivation conditions. In the isoprenoid-producing plant of the present invention, the polyisoprenoid biosynthetic pathway is suitably enhanced by enhancing the rate-limiting site of IPP biosynthesis by the MVA pathway by the introduced bHLH transcription factor. The production amount of can be increased suitably.

In this specification, polyisoprenoids is a general term for structured polymer with isoprene units (C 5 _{H 8).} Examples of polyisoprenoids include monoterpenes (C ₁₀ ), sesquiterpenes (C ₁₅ ), diterpenes (C ₂₀ ), sesterterpenes (C ₂₅ ), triterpenes (C ₃₀ ), tetraterpenes (C ₄₀ ), and natural rubber. A polymer is mentioned.

As described above, in the present invention, the expression of hydroxymethylglutaryl CoA reductase can be regulated (enhanced) by the bHLH type transcription factor, so that the rate limiting site of IPP biosynthesis by the MVA pathway is enhanced, so that polyisoprenoid The biosynthetic pathway is suitably enhanced, and the amount of polyisoprenoid produced in the transformant can be suitably increased.

Further, the isoprenoid-producing plant of the present invention into which a gene encoding a bHLH transcription factor has been introduced has an enhanced rate-limiting site for IPP biosynthesis by the MVA pathway by the introduced bHLH transcription factor. Since the biosynthetic pathway is suitably enhanced, the production amount of polyisoprenoid can be suitably increased by producing polyisoprenoid using the isoprenoid-producing plant.

As described above, since the production amount of polyisoprenoid can be increased by the method of the present invention, the isoprenoid-producing plant of the present invention, and the polyisoprenoid production method of the present invention, it is possible to cope with the depletion of natural rubber resources that are concerned. It is possible.

The present invention will be specifically described based on examples, but the present invention is not limited to these examples.

(Promoter sequence preparation)
A gene encoding HMG-CoA reductase derived from the leaf of Para rubber tree (the base sequence of the gene is shown in SEQ ID NO: 7 in the sequence listing) and a DNA fragment containing the promoter were cloned by the following procedure. First, genomic DNA was extracted from the leaves of Para rubber tree. Extraction was performed using a commercially available genomic DNA extraction kit. Amplification of the gene containing the promoter region of the gene encoding HMG-CoA reductase is performed by TAIL-PCR using a random primer as shown in primers 1 to 6 and a primer corresponding to the gene encoding HMG-CoA reductase. Obtained.
Primer 1: 5′-ntcgastwtsgwgtt-3 ′ (SEQ ID NO: 9)
Primer 2: 5′-nggtcgtswganawgaa-3 ′ (SEQ ID NO: 10)
Primer 3: 5′-wgtgnagwancanag-3 ′ (SEQ ID NO: 11)
Primer 4: 5′-sttntastntntgc-3 ′ (SEQ ID NO: 12)
Primer 5: 5′-sstggstanwatwatt-3 ′ (SEQ ID NO: 13)
Primer 6: 5′-agwgnagwancanaga-3 ′ (SEQ ID NO: 14)

As a result of examining the base sequence of the DNA fragment obtained using the above primer, the promoter sequence of HMG-CoA reductase was obtained. The base sequence of the promoter sequence of HMG-CoA reductase is shown in SEQ ID NO: 15.

The promoter sequence was analyzed using an analysis database for plant promoters (A Date of Of Cis-Regulating DNA Elements (PLACE)) (http://www.dna.affrc.go.jp/PLACE/).

As a result of the analysis, many binding (recognition) sites (CANNTG) of bHLH type transcription factors were found.

(Amplification of promoter region)
The promoter region of the above gene was amplified by PCR in the following range.
HMG-CoA reductase promoter: −1 to −1500 bp, −1 to −1000 bp, −1 to −500 bp
The PCR product was cloned into pMD20T (manufactured by Takara Bio Inc.) to construct pMD20T-hmgpro (-1500), pMD20T-hmgpro (-1000), and pMD20T-hmgpro (-500). It was confirmed that there was no mutagenesis by conducting a sequence analysis of the inserted PCR product.

(Construction of reporter sequence binding vector)
The plasmid constructed in (amplification of promoter region) is restricted with SpeI and HindIII, KpnI, or BamHI, so that the site where the T7 promoter region of pYES3 / CT / LacZ is removed, ie, immediately above the reporter lacZ gene. Each promoter sequence fragment was incorporated into pYES3-hmgprolacZ (-1500), pYES3-hmgprolacZ (-1000), and pYES3-hmgpro (-500). Ligation is performed using Ligation high ver. 2 (manufactured by TOYOBO).

(Construction of gene transfer vector into yeast chromosome)
Amplification of the plasmid from the SpeI site of the plasmid constructed in (construction of reporter sequence binding vector) to CYC1 transcription termination signal by PCR, and the restriction of the fragments with SalI, SmaI, XbaI, or SphI allows various promoter sequences. And a lacZ gene ligated DNA fragment. In order to insert the obtained DNA fragment on the chromosome of yeast, it was incorporated into pAUR101 DNA (manufactured by Takara Bio Inc.) which was also treated with restriction enzymes, and pAUR101-hmgprolacZ (-1500), pAUR101-hmgprolacZ (-1000), pAUR101-hmgpro (-500) was constructed. Ligation was performed in the same way as in Ligation high ver. 2 was used.

(Acquisition of transcription factor gene)
Next, PCR was performed using an Arabidopsis cDNA library as a template. Three types of PCR fragments, At1g63650 (Myc2) (SEQ ID NO: 1), At5g54680 (ILR3) (SEQ ID NO: 3), and At1g02340 (HFR1) (SEQ ID NO: 5) were obtained by PCR. The obtained PCR products were cloned into pMD20T to construct pMD20T-Myc2, pMD20T-ILR3, and pMD20T-HFR1. It was confirmed that there was no mutagenesis by conducting a sequence analysis of the inserted PCR product.

(Construction of transcription factor expression vector)
By restricting the plasmid constructed in (Acquisition of transcription factor gene) with SpeI, BamHI, or EcoRV, each transcription factor gene is incorporated downstream of the TEF1 promoter region of p427TEF (manufactured by Cosmo Bio), and pTEF-Myc2 PTEF-ILR3 and pTEF-HFR1 were constructed. Ligation is performed using Ligation high ver. 2 was used.

(Yeast transformation)
The plasmid constructed in (Construction of gene introduction vector into yeast chromosome) and (Construction of transcription factor expression vector) was introduced into yeast (BY4741 strain) by electroporation. Selection of the transgenic yeast was performed by culturing the yeast on a medium containing Aureobasidin A (Takara Bio) and G418 (Wako Pure Chemical Industries), which are antifungal antibiotics.

(Confirmation of transcription factor effect)
The transformed yeast was cultured on a medium containing X-gal to investigate the expression of lacZ due to transcription factor activity. Specifically, when lacZ, which is a reporter gene linked to a promoter sequence, is expressed, X-gal contained in the medium is decomposed to exhibit a blue color. Therefore, when blue is displayed, lacZ is expressed due to transcription factor activity. It was judged. Table 1 shows the number of times lacZ was expressed by transcription factor activity after 10 tests.

From Table 1, use of At1g63650 (Myc2) (SEQ ID NO: 1, 2), At5g54680 (ILR3) (SEQ ID NO: 3, 4), At1g02340 (HFR1) (SEQ ID NO: 5, 6) (particularly At1g63650 (Myc2)) It was found that the activity of the reporter gene increased. Furthermore, the longer the promoter region sequence, the greater the number of times that the reporter gene activity was expressed, that is, the greater the number of binding sites of At1g63650 (Myc2), At5g54680 (ILR3), and At1g02340 (HFR1), the greater the promoter sequence. Since At1g63650 (Myc2), At5g54680 (ILR3), and At1g02340 (HFR1) functioned as transcription factors of HMG-CoA reductase, it was proved. Therefore, by introducing At1g63650 (Myc2), At5g54680 (ILR3), At1g02340 (HFR1) (especially At1g63650 (Myc2)) into the isoprenoid-producing plant, the rate-limiting part of IPP biosynthesis by the MVA pathway is enhanced, and polyisoprenoid It was proved that the biosynthetic pathway can be suitably enhanced and the production of polyisoprenoid can be suitably increased.

(Sequence table free text)
SEQ ID NO: 1: base sequence of a gene encoding Myc2 derived from Arabidopsis SEQ ID NO: 2: amino acid sequence of Myc2 derived from Arabidopsis SEQ ID NO: 3: base sequence of a gene encoding ILR3 derived from Arabidopsis SEQ ID NO: 4: of ILR3 derived from Arabidopsis Amino acid sequence SEQ ID NO: 5: base sequence of gene encoding HFR1 derived from Arabidopsis thaliana SEQ ID NO: 6: amino acid sequence of HFR1 derived from Arabidopsis thaliana SEQ ID NO: 7: base sequence of gene encoding HMG-CoA reductase derived from Para rubber tree Amino acid sequence of HMG-CoA reductase derived from Para rubber tree SEQ ID NO: 9: Primer 1
Sequence number 10: Primer 2
Sequence number 11: Primer 3
Sequence number 12: Primer 4
Sequence number 13: Primer 5
SEQ ID NO: 14: Primer 6
SEQ ID NO: 15: Base sequence of promoter sequence of HMG-CoA reductase derived from Para rubber tree

Claims (4)

b asic-helix-loop-helix type transcription factor genes encoding (bHLH transcription factor) is introduced into a host, the host, enhance the expression of HMG CoA reductase,
The gene is DNA described in [1] or [2] below,
Host is, Hevea spp, Sonchus spp., Taraxacum spp., And Ru plant der belonging to at least one of the genus selected from the group consisting of Parthenium genus methods.
[1] DNA comprising the base sequence represented by any one of SEQ ID NOs: 1, 3 and 5
[2] DNA encoding a protein comprising a base sequence having 90% or more sequence identity with the base sequence represented by any one of SEQ ID NOs: 1, 3, and 5 and having transcription factor activity The method according to claim 1 , wherein the bHLH transcription factor is a protein according to any one of [1] to [3] below.
[1] A protein comprising the amino acid sequence represented by SEQ ID NO: 2, 4, or 6 [2] Substitution or deletion of 1 to 60 amino acids in the amino acid sequence represented by SEQ ID NO: 2 A protein having a transcription factor activity and comprising the sequence comprising insertion, insertion, and / or addition, substitution, deletion, insertion, and / or addition of 1 to 23 amino acids in the amino acid sequence represented by SEQ ID NO: 4 A protein having a transcription factor activity, or a sequence comprising substitution, deletion, insertion and / or addition of 1 to 29 amino acids in the amino acid sequence represented by SEQ ID NO: 6 and it consists proteins [3] one of SEQ ID represented by the amino acid sequence and amino acid sequence having 90% or more sequence identity with SEQ ID NO: 2, 4, 6 having a transfer factor activity, and transcription factors Protein having an activity a gene encoding a bHLH transcription factor was introduced ,
The gene is DNA described in [1] or [2] below,
An isoprenoid-producing plant that is a plant belonging to at least one genus selected from the group consisting of the genus Hevea, Sonchus, Taraxacum, and Parthenium .
[1] DNA comprising the base sequence represented by any one of SEQ ID NOs: 1, 3 and 5
[2] DNA encoding a protein comprising a base sequence having 90% or more sequence identity with the base sequence represented by any one of SEQ ID NOs: 1, 3, and 5 and having transcription factor activity A method for producing a polyisoprenoid using the isoprenoid-producing plant according to claim 3 .

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